The high affinity receptor for IgE (FceRI) is central to allergic reactions. It is a tetrameric structure (a[3y2) when expressed on mast cells and basophils. The c_ chain is solely responsible for binding the ligand IgE while the [3 and 7 chains are involved in signaling. Aggregation of Fc_RI by IgE and the corresponding multivalent antigen induces the release of allergy mediators and proinflammatory cytokines. One of the critical events that connect Fc_RI aggregation with mediator and cytokine secretion is Ca z+ mobilization. Ca z mobilization is comprised of Ca 2 release from intracellular stores, and Ca 2 entry from the extracellular milieu. The role played by phospholipase Cy in contributing to Ca 2 release from intracellular stores is welt documented. It produces the second messenger 1,4,5-triphosphate (IP3) that binds to IP 3 receptors, thereby inducing Ca 2+ release from intracellular stores. A few years ago we showed that, in addition to the well- known role played by phospholipase Cy, sphingosine 1-phosphate (S 1P) was critical in Ca 2+ release induced by FcERI. S1P is produced by the action of a sphingosine kinase activity on sphingosine. Two sphingosine kinases have been cloned but they have not been shown to be involved in the Ca 2+ release induced by Fc_RI aggregation. In this proposal our central hypothesis is that Fc_RI-dependent Ca 2+ release requires both PLCy and PI3KC21_ activation and that PI3KC21_ plays a major role in mast cell function. We present preliminary data showing that PI3KC213 possesses SK activity, is activated after FcERI aggregation, and contributes significantly to Ca z release. In Aim 1 we will elucidate the mechanisms of FceRI-mediated activation of the PI3KC213 sphingosine kinase activity. In Aim 2 we will dissect the structural requirements underlying the sphingosine kinase function of PI3KC213.In Aim 3 we will elucidate the role of sphingosine kinase function of PI3KC213 in mast cell development and mast cell function in vitro and in vivo using mice with a mast cell specific PI3KC213 deficiency. All together, the proposed experiments will provide a comprehensive characterization of PI3KC213 and its sphingosine kinase activity, and define its role in mast cell physiology.